發(fā)布時(shí)間：2018-06-21 15:15

  本文選題：骨形態(tài)蛋白-2 + 腺病毒��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：背景和目的:各種原因造成的骨組織缺損是臨床常見(jiàn)疾患,隨著骨組織工程技術(shù)的興起,給傳統(tǒng)治療難點(diǎn)的骨缺損帶來(lái)了一些曙光,組織工程利用自體細(xì)胞的生物活性,給予外在因素特定的干擾下將其誘導(dǎo)分化為所需的細(xì)胞,從而達(dá)到修復(fù)組織的能力。傳統(tǒng)的間充質(zhì)干細(xì)胞、胚胎干細(xì)胞已在體外成功被誘導(dǎo)定向分化,但涉及到一些倫理、取材限制等問(wèn)題,因此其在組織學(xué)上作為種子方面的研究受到一定的限制;而隨著體內(nèi)體細(xì)胞的研究,通過(guò)使用幾個(gè)特定的轉(zhuǎn)錄因子誘導(dǎo)體細(xì)胞的重編程而獲得的可不斷自我更新且具有多向分化潛能的誘導(dǎo)性多能干細(xì)胞(induced pluripotent stem cells,i PSCs)的出現(xiàn),可作為組織學(xué)中理想的種子細(xì)胞,本課題在前期已成功將人、山羊、大鼠成纖維細(xì)胞誘導(dǎo)為i PSCs的基礎(chǔ)上,運(yùn)用誘導(dǎo)因子將山羊i PSCs定向分化為i PSCs-MSC,作為骨組織工程的優(yōu)化種子細(xì)胞,然后利用體外構(gòu)建腺病毒搭載生物因子BMP-2,從而去轉(zhuǎn)染誘導(dǎo)i PSCs-MSC,探討載體介導(dǎo)下種子細(xì)胞轉(zhuǎn)染的可行性,同時(shí)觀察研究i PSCs-MSC在生物因子BMP-2的作用下,對(duì)其體外增殖和分化成骨能力的影響。方法:1.體外擴(kuò)增預(yù)先構(gòu)建好的i PSCs-MSC,取傳代至第三代的i PSCs-MSC,按照普通培養(yǎng)基、含BMP-2的培養(yǎng)基、腺病毒-BMP-2的培養(yǎng)基對(duì)其進(jìn)行體外繁殖,而分別設(shè)定為空白對(duì)照組、陰性對(duì)照組和實(shí)驗(yàn)組,然后分別往各組加入成骨誘導(dǎo)液,在體外進(jìn)行培養(yǎng),分別在培養(yǎng)1、2、3、4、5、6、7d用CCK-8試劑檢測(cè)i PSCs-MSC的存活及增殖活性;2.三組處理好的細(xì)胞,轉(zhuǎn)染后,分別于3、7、14天后終止培養(yǎng),利用堿性磷酸酶試劑盒檢測(cè)其堿性磷酸酶表達(dá)情況;3.然后在三組細(xì)胞培養(yǎng)增殖至20天后,使用茜素紅染液,然后分別于染色后1周、2周后用光學(xué)顯微鏡下觀察礦化結(jié)節(jié)的形成;4.使用Western Blotting法測(cè)定三組細(xì)胞,檢測(cè)轉(zhuǎn)染后BMP-2的表達(dá)情況。結(jié)果:1.CCK-8檢測(cè)后,實(shí)驗(yàn)組細(xì)胞的可見(jiàn)光吸收度OD高于另外兩項(xiàng)對(duì)照組,說(shuō)明實(shí)驗(yàn)組中細(xì)胞增殖能力活性明顯強(qiáng)于另外兩組;2.腺病毒轉(zhuǎn)染后的三組細(xì)胞ALP測(cè)定呈陽(yáng)性反應(yīng),且細(xì)胞內(nèi)ALP活性隨著時(shí)間的延長(zhǎng)而增加,但是實(shí)驗(yàn)組中ALP含量明顯高于另外兩組,同時(shí)隨著時(shí)間延長(zhǎng),陰性對(duì)照組較空白對(duì)照組也表現(xiàn)出強(qiáng)ALP表達(dá)能力;3.實(shí)驗(yàn)組紅色礦化結(jié)節(jié)含量明顯,陰性對(duì)照組礦化結(jié)節(jié)表達(dá)稍高于空白對(duì)照組,空白對(duì)照組未見(jiàn)明顯紅色結(jié)節(jié);4.各組細(xì)胞轉(zhuǎn)染后,實(shí)驗(yàn)組細(xì)胞BMP-2表達(dá)明顯高于陰性對(duì)照組,陰性對(duì)照組表達(dá)不明顯,空白對(duì)照組未見(jiàn)表達(dá)。結(jié)論:1.證實(shí)了i PSC-MSCs在體外成骨的可行性;2.說(shuō)明了體外生物因子BMP-2具有能顯著促進(jìn)i PSC-MSCs向成骨細(xì)胞的分化和功能;3.腺病毒能明顯加快體外BMP-2對(duì)i PSC-MSCs向成骨細(xì)胞的分化速率;4.BMP-2基因轉(zhuǎn)染誘導(dǎo)性多能間充質(zhì)干細(xì)胞后在體外可明顯促進(jìn)其向成骨細(xì)胞分化,并為其作為骨組織工程優(yōu)化種子細(xì)胞奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Background and objective: bone tissue defect caused by various causes is a common clinical disease. With the rise of bone tissue engineering technology, it brings some light to the traditional treatment of bone defect. Tissue engineering makes use of the biological activity of autosomal cells. The ability of tissue repair can be achieved by inducing the cells to differentiate into the required cells under the specific interference of external factors. Traditional mesenchymal stem cells, embryonic stem cells have been successfully induced directional differentiation in vitro, but related to a number of ethical, material constraints, so the study of histology as a seed is limited to a certain extent; With the study of somatic cells in vivo, induced pluripotent stem cells induced by inducing pluripotent stem cells, which can be self-renewing and have multi-differentiation potential, were obtained by reprogramming several specific transcription factor inducers. It can be used as an ideal seed cell in histology. We have successfully induced human, goat and rat fibroblasts into I PSCs in the early stage. Goat I-PSCs were differentiated into I-PSCs-MSCs by inducing factors, which were used as seed cells for bone tissue engineering. Then, the adenovirus was constructed to carry the biological factor BMP-2 in vitro, which was used to transfect and induce iPSCs-MSC.To explore the feasibility of vector mediated seed cell transfection, and to observe the effect of iPSCs-MSC on the biological factor BMP-2. Effects on proliferation and osteogenic differentiation in vitro. Method 1: 1. The pre-constructed I PSCs-MSCs were amplified in vitro, and then passed to the third generation I PSCs-MSC.According to the normal medium, the medium containing BMP-2 and the medium of adenovirus BMP-2, they were propagated in vitro, and were set as blank control group, negative control group and experimental group, respectively. Then the osteoblasts were added to each group and cultured in vitro. The survival and proliferative activity of iPSCs-MSC were detected by CCK-8 reagent on the 7th day of culture. The three groups of treated cells were transfected, and the culture was terminated after 14 days after transfection. The alkaline phosphatase expression was detected by alkaline phosphatase kit. The cells in the three groups were cultured for 20 days and then stained with alizarin red. The formation of mineralized nodules was observed under optical microscope at 1 week and 2 weeks after staining. The expression of BMP-2 was detected by Western blotting. Results 1. The OD of the cells in the experimental group was higher than that in the other two control groups after CCK-8 detection, which indicated that the proliferative activity of the cells in the experimental group was significantly higher than that in the other two groups. The ALP activity of the three groups after adenovirus transfection increased with time, but the ALP content in the experimental group was significantly higher than that in the other two groups, and the ALP activity increased with time. The negative control group also showed stronger ALP expression ability than the blank control group. The content of red mineralized nodules in the experimental group was obvious, the expression of the mineralized nodules in the negative control group was slightly higher than that in the blank control group, and no obvious red nodule was found in the blank control group. After transfection, the expression of BMP-2 in the experimental group was significantly higher than that in the negative control group, but not in the blank control group. Conclusion 1. The feasibility of I PSC-MSCs osteogenesis in vitro was confirmed. The results showed that BMP-2 could significantly promote the differentiation and function of I PSC-MSCs into osteoblasts in vitro. Adenovirus could significantly accelerate the differentiation rate of I PSC-MSCs into osteoblasts in vitro. 4. BMP-2 gene transfection into induced pluripotent mesenchymal stem cells could obviously promote the differentiation of I PSC-MSCs into osteoblasts in vitro. It also lays the experimental foundation for the optimization of seed cells in bone tissue engineering.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R68

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相關(guān)期刊論文 前2條

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本文編號(hào)：2049197